JP2021007417A - Biosynthesis of human milk oligosaccharides in engineered bacteria - Google Patents

Abstract

To provide compositions and methods for engineering bacteria to produce fucosylated oligosaccharide, and uses thereof in prevention or treatment of infections.SOLUTION: The invention provides a method for producing fucosylated oligosaccharide in a bacterium comprising the steps of: providing a bacterium comprising a functional β-galactosidase gene, an exogenous fucosyl transferase gene, a GDP-fucose synthesis pathway and a functional lactose permease gene; culturing the bacterium in the presence of lactose; and retrieving a fucosylated oligosaccharide from a culture supernatant of the bacterium.SELECTED DRAWING: Figure 3

Description

Related Applications This application is prioritized over US Patent Provisional Application No. 61 / 443,470 filed February 16, 2011, which is incorporated herein by reference in its entirety under 35 USC § 119 (e). Claim the benefit of rights.

Fields of Invention The present invention provides compositions and methods for producing purified oligosaccharides, particularly certain fucosylated and / or sialylated oligosaccharides typically found in human milk.

Background of the Invention Human milk contains a diverse and abundant series of neutral and acidic oligosaccharides (human milk oligosaccharides, HMOS). Many of these molecules are not directly utilized as nutrition by infants, but play important roles in establishing healthy intestinal microbiomes, disease prevention, and immune function. Prior to the present invention described herein, there was a problem with the ability to produce HMOS on a large scale and at low cost. For example, HMOS production by chemical synthesis was limited by stereospecificity issues, precursor availability, product impurities, and generally high costs. Therefore, there is an urgent need for new strategies to inexpensively produce large quantities of HMOSs for a variety of commercial applications.

The invention described herein features an efficient and economical method for producing fucosylated and sialylated oligosaccharides. Methods of producing fucosylated oligosaccharides in bacteria include the following steps: Bacteria containing a functional β-galactosidase gene, an exogenous fucosyl transferase gene, a GDP-fucose synthetic pathway, and a functional lactose permease gene. The step of providing; the step of culturing the bacterium in the presence of lactose; and the step of recovering the fucosylated oligosaccharide from the bacterium or from the bacterial culture supernatant.

To produce fucosylated oligosaccharides by biosynthesis, bacteria utilize the endogenous or extrinsic guanosine diphosphate (GDP) -fucose synthetic pathway. By "GDP-fucose synthetic pathway" is meant a series of reactions in which GDP-fucose is synthesized, usually controlled and catalyzed by enzymes. An exemplary GDP-fucose synthetic pathway in Escherichia coli is described below. In the GDP-fucose synthesis pathway described below, the enzymes for GDP-fucose synthesis are 1) manA = phosphomannose isomerase (PMI), 2) manB = phosphomannomutase (PMM), 3) manC = mannose. -1-guanylyl phosphate transferase (GMP), 4) gmd = GDP-mannose-4,6-dehydrase (GMD), 5) fcl = GDP-fucose synthase (GFS), and 6) ΔwcaJ = mutant UDP- Includes glucose lipid carrier transferase.

The synthetic pathway from fructose-6-phosphate to GDP-fucose, a common metabolic intermediate in all organisms, consists of five enzymatic steps: 1) PMI (phosphomannose isomerase), 2) PMM (phosphomannomutase). Nomutase), 3) GMP (mannose-1-phosphate guanylyl transferase), 4) GMD (GDP-mannose-4,6-dehydrase), and 5) GFS (GDP-fucose synthase). Individual bacterial species differ in their inherent ability to synthesize GDP-fucose. For example, E. coli contains enzymes that are competent to perform all five stages, while Bacillus licheniformis has enzymes that can perform stages 4 and 5 (ie, GMD and GFS). It is missing. Any enzyme in the GDP synthetic pathway that is inherently deleted in any particular bacterial species is provided as a gene in a recombinant DNA construct and either in a plasmid expression vector or as an exogenous gene incorporated into the host chromosome. Supplied.

The present invention described herein relates to the manipulation of genes and pathways within bacteria such as the gut flora Escherichia coli K12 (E. coli) or probiotic bacteria, which results in high levels of HMOS synthesis. Describe in detail. Diverse bacterial species, such as Erwinia herbicola, Pantoea agglomerans, Citrobacter freundii, Pantoea citrea, Pectobacterium carotobolum , Or Xanthomonas campestris can be used in the oligosaccharide biosynthesis method. Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus laterosporus, Bacillus mydesporus, Bacillus megaterium ), Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus bacteria, including Bacillus circulans, can be used as well. Similarly, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus helveticus Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus casei Lactobacillus casei Lactobacillus casei (Lactobacillus reuteri), Lactobacillus jensenii, and Lactobacillus lactis, but not limited to, the bacteria of the genus Lactobacillus and Lactococcus. It can be modified using the method of the invention. Streptococcus thermophiles and Propionibacterium freudenreichii are also suitable bacterial species for the present invention as described herein. Enterococcus (eg, Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (eg, Bifidobacterium longum, Bifidobacterium), Bifidobacterium Bifidobacterium infantis, and Bifidobacterium bifidum, Sporolactobacillus, Micromomospora, Micrococcus, Rodococcus (Bifidobacterium infantis), and Bifidobacterium bifidum. Rhodococcus) species and strains of Pseudomonas fluorescens (eg, Pseudomonas fluorescens and Pseudomonas aeruginosa) are also included as part of the present invention modified as described herein. .. Bacteria containing the characteristics described herein are cultured in the presence of lactose, and fucosylated oligosaccharides are recovered from either the bacteria themselves or the bacterial culture supernatant. Fucosylated oligosaccharides are purified for use in therapeutic formulations or nutritional products, or bacteria are used directly in such products.

Bacteria also contain the functional β-galactosidase gene. The β-galactosidase gene is either an endogenous β-galactosidase gene or an extrinsic β-galactosidase gene. For example, the β-galactosidase gene includes the E. coli lacZ gene (eg, GenBank Accession No. V00296 (GI: 41901), incorporated herein by reference). Bacteria accumulate increased intracellular lactose spools to produce low levels of β-galactosidase.

The functional lactose permease gene is also present in bacteria. The lactose permase gene is either an endogenous lactose permase gene or an extrinsic lactose permase gene. For example, the lactose permease gene includes the E. coli lacY gene (eg, GenBank Accession No. V00295 (GI: 41897), incorporated herein by reference). Many bacteria are found in homologues of E. coli lactose permease (eg, as found in Bacillus likeniformis) or in ubiquitous PTS sugar transporter family members (eg, Lactobacillus casei and Lactobacillus ramnosus). By utilizing the transporter protein, which is one of the transporters, it inherently possesses the ability to transport lactose from the growth medium to the cells. For bacteria that are inherently deficient in the ability to transport extracellular plasmids into the cytoplasm of the cell, this ability is conferred by the exogenous lactose transporter gene provided in the recombinant DNA construct (eg, E. coli lacY). It is supplied either on a plasmid expression vector or as an exogenous gene integrated into the host chromosome.

Bacteria contain an exogenous fucosyltransferase gene. For example, the exogenous fucosyltransferase gene encodes α (1,2) fucosyltransferase and / or α (1,3) fucosyltransferase. An exemplary α (1,2) fucosyltransferase gene is the wcfW gene (SEQ ID NO: 4) of Bacteroides fragilis NCTC 9343. An exemplary α (1,3) fucosyltransferase gene is the Helicobacter pylori 26695 futA gene. An example of the Helicobacter pylori futA gene is represented in GenBank Accession No. HV532291 (GI: 365791177), which is incorporated herein by reference.

Alternatively, methods of producing fucosylated oligosaccharides by biosynthesis include the following steps: functional β-galactosidase gene, extrinsic fucosyl transferase gene, mutations in cholanoic acid synthesis gene, and functional lactose permease gene. The step of providing enterobacteria containing; the step of culturing the bacterium in the presence of lactose; and the step of recovering the fucosylated oligosaccharide from the bacterium or the culture supernatant of the bacterium.

To produce fucosylated oligosaccharides by biosynthesis, the bacterium contains mutations in the endogenous koranic acid (fucose-containing exopolysaccharide) synthetic gene. By "cholanoic acid synthase gene" is meant a gene involved in a series of reactions in which cholanoic acid synthesis occurs, usually controlled and catalyzed by an enzyme. Exemplary colacic acid synthesis genes include the rcsA gene (eg, GenBank accession number M58003 (GI: 1103316), incorporated herein by reference), the rcsB gene (eg, GenBank accession number E04821 (GI: 2173017)). ), WcaJ gene (eg, GenBank accession number (amino acid) BAA15900 (GI: 1736749), incorporated herein by reference), wzxC gene (eg, GenBank accession number). (Amino acid) BAA15899 (GI: 1736748), incorporated herein by reference), wcaD gene (eg, GenBank accession number (amino acid) BAE76573 (GI: 85675202), incorporated herein by reference), wza Genes (eg, GenBank Accession Number (amino acid) BAE76576 (GI: 85675205), incorporated herein by reference), wzb gene (eg, GenBank Accession Number (amino acid) BAE76575 (GI: 85675204), reference to the book. Included herein), and the wzc gene (eg, GenBank accession number (amino acid) BAA15913 (GI: 1737663), incorporated herein by reference).

It is obtained through a number of genetic modifications of the endogenous E. coli gene that are either directly involved in colanic acid precursor biosynthesis or involved in the overall regulation of colanate synthetic regulons. Specifically, the ability of the host E. coli strain to synthesize the extracellular capsular polysaccharide colanic acid is abolished by the deletion of the wcaJ gene, which encodes the UDP-glucose lipid carrier transferase. In the wcaJ null background, GDP-fucose accumulates in the cytoplasm of E. coli. Overexpression of RcsA, a positive regulatory protein in the cholanoic acid synthesis pathway, results in increased intracellular GDP-fucose levels. A further positive regulator of colanic acid biosynthesis, namely RcsB overexpression, is also utilized in place of or in addition to RcsA overexpression to increase intracellular GDP-fucose levels. .. Alternatively, colanate biosynthesis is increased after introducing a null mutation into the E. coli lon gene (eg, GenBank Accession No. L20572 (GI: 304907), incorporated herein by reference). Lon is an adenosine-5'-triphosphate (ATP) -dependent intracellular protease that causes the above-mentioned RcsA degradation as a positive transcriptional regulator of cholanic acid biosynthesis in Escherichia coli. In lon null background, RcsA is stabilized, RcsA levels are increased, genes responsible for GDP-fucose synthesis in E. coli are upregulated, and intracellular GDP-fucose levels are enhanced.

For example, the bacterium also contains an endogenous gene, such as the functional wild-type E. coli lacZ ⁺ gene inserted into the E. coli lon gene. In this way, the bacterium can contain mutations in the lon gene.

Bacteria also contain the functional β-galactosidase gene. The β-galactosidase gene is either an endogenous β-galactosidase gene or an extrinsic β-galactosidase gene. For example, the β-galactosidase gene contains the E. coli lacZ gene. The E. coli endogenous lacZ gene is either deleted or functionally inactivated such that expression of the downstream lactose permease (lacY) gene remains intact.

Bacteria contain an exogenous fucosyltransferase gene. For example, the exogenous fucosyltransferase gene encodes α (1,2) fucosyltransferase and / or α (1,3) fucosyltransferase. An exemplary α (1,2) fucosyltransferase gene is the wcfW gene (SEQ ID NO: 4) of Bacteroides fragilis NCTC 9343. An exemplary α (1,3) fucosyltransferase gene is the Helicobacter pylori 26695 futA gene. An example of the Helicobacter pylori futA gene is represented in GenBank Accession No. HV532291 (GI: 365791177), which is incorporated herein by reference.

The functional lactose permease gene is also present in bacteria. The lactose permase gene is either an endogenous lactose permase gene or an extrinsic lactose permase gene. For example, the lactose permease gene contains the E. coli lacY gene.

Bacteria can further contain the exogenous rcsA and / or rcsB gene (eg, in an ectopic nucleic acid construct such as a plasmid), and the bacterium can optionally include the lacA gene (eg, GenBank accession number X51872 (GI:)). 41891), which is incorporated herein by reference) further.

Bacteria containing the characteristics described herein are cultured in the presence of lactose to recover fucosylated oligosaccharides from the bacteria themselves or from the bacterial culture supernatant. Fucosylated oligosaccharides are purified for use in therapeutic formulations or nutritional products, or bacteria are used directly in such products.

Bacteria used herein to produce HMOS include increased intracellular guanosine diphosphate (GDP) -fucose pool, increased intracellular lactose spool (compared to wild type), and It is genetically engineered to include fucosyl transferase activity. Thus, bacteria contain mutations in colanic acid (fucose-containing exopolysaccharide) synthesis pathway genes such as the wcaJ gene, which results in enhancement of intracellular GDP-fucose pool. Bacteria also contain an endogenous gene, such as the functional wild-type E. coli lacZ ⁺ gene inserted into the E. coli lon gene. In this way, the bacterium can also contain mutations in the lon gene. The E. coli endogenous lacZ gene is either deleted or functionally inactivated such that expression of the downstream lactose permease (lacY) gene remains intact. Organisms so engineered have the ability to transport lactose from growth media, as well as acceptor sugars in oligosaccharide synthesis, while maintaining low levels of intracellular β-galactosidase activity that are useful for a variety of additional purposes. Maintain the ability to make intracellular lactose spools for use. Bacteria can further contain exogenous rcsA and / or rcsB genes (eg, in ectopic nucleic acid constructs such as plasmids), and bacteria optionally further mutate in the lacA gene. Preferably, the bacterium accumulates an increased intracellular lactose spool to produce low levels of β-galactosidase.

Bacteria possess fucosyltransferase activity. For example, the bacterium contains one or both of an exogenous fucosyltransferase gene encoding α (1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α (1,3) fucosyltransferase. An exemplary α (1,2) fucosyltransferase gene is the wcfW gene (SEQ ID NO: 4) of Bacteroides fragilis NCTC 9343. Prior to the present invention, this wcfW gene was not known to encode a protein having α (1,2) fucosyltransferase activity, and was thought to possess the ability to utilize lactose as an acceptor sugar. There wasn't. Other α (1,2) fucosyltransferase genes that use lactose as the acceptor sugar (eg, the Helicobacter pylori 26695 futC gene, or the E. coli O128: B12 wbsJ gene) can be easily replaced with Bacteroides fragilis wcfW. An example of the Helicobacter pylori futC gene is represented in GenBank Accession No. EF452503 (GI: 134142866), which is incorporated herein by reference.

An exemplary α (1,3) fucosyltransferase gene is the Helicobacter pylori 26695 futA gene, but other α (1,3) fucosyltransferase genes known in the art (eg, Helicobacter helicobacter). It may be replaced with hepaticus) Hh0072, Helicobacter bilis, Campylobacter jejuni, or α (1,3) fucosyltransferase gene from Bacteroides species). The present invention includes nucleic acid constructs containing one, two, three, or more of the above genes. For example, the invention presents a deleted endogenous β-galactosidase (eg, lacZ) gene, a low-activity replacement functional β-galactosidase gene, a GDP-fucose synthetic pathway, a functional lactose permease gene, and a deletion. A nucleic acid construct expressing an exogenous fucosyltransferase gene (encoding α (1,2) fucosyltransferase or α (1,3) fucosyltransferase) transformed into a bacterial host strain containing the lactose acetyltransferase gene. Including.

Similarly, the isolated E. coli bacteria described above are characterized to contain a defective cholanoic acid synthesis pathway, reduced levels of β-galactosidase (LacZ) activity, and an exogenous fucosyltransferase gene. Included in the invention. The invention also a) utilizes β-galactosidase (eg, lacZ) gene inserts engineered to produce low but readily detectable levels of β-galactosidase activity in β-galactosidase-negative host cells. A method for phenotypic labeling of loci, b) a method for easily detecting lysed bacteriophage contamination during fermentation execution through release and detection of cytoplasmic β-galactosidase in a cell culture medium, and c) a method for bacterially detecting residual lactose at the end of production execution Each of a), b), and c) was carefully engineered to induce low but detectable levels of β-galactosidase activity in otherwise β-galactosidase-negative host cells, including methods of depletion from culture. This is done by utilizing a functional β-galactosidase (eg, lacZ) gene insert.

Purified fucosylated oligosaccharides produced by the above method are also within the scope of the present invention. Purified oligosaccharides such as 2'-FL, 3FL, LDFT are oligos in which the desired oligosaccharide is at least 90% by weight, 95% by weight, 98% by weight, 99% by weight, or 100% by weight (w / w). It is sugar. Purity is assessed by any known method, such as thin layer chromatography or other electrophoresis or chromatography techniques known in the art. The present invention comprises the steps of separating the desired fucosylated oligosaccharide (eg, 2'-FL) from a bacterial cell extract or lysate, or contaminant in bacterial cell culture supernatant, by the genetically engineered bacteria described above. Includes a method of purifying the fucosylated oligosaccharides produced. Contaminations include bacterial DNA, proteins and cell wall components, and sometimes yellow / brown sugar caramel formed by spontaneous chemical reactions in culture medium.

Oligosaccharides are purified and used in numerous products for consumption by animals such as humans and companion animals (dogs, cats) and livestock (cattle, horses, sheep, goats, or poultry with pigs). For example, purified 2'-fucosyl lactose (2'-FL), 3-fucosyl lactose (3FL), lactodifucotetraose (LDFT), or 3'-sialyl-3-fucosyl lactose (3'-S3FL), and Pharmaceutical compositions containing excipients are suitable for oral administration. Large amounts of 2'-FL, 3FL, LDFT, or 3'-S3FL are bacterial hosts such as heterologous α (1,2) fucosyltransferase, heterologous α (1,3) fucosyltransferase, or heterologous sialyltransferase, or mixtures thereof. Produced in Escherichia coli bacteria, including. E. coli bacteria containing enhanced cytoplasmic pools of lactose, GDP-fucose, and CMP-Neu5Ac are useful in such production systems. In the case of lactose and GDP-fucose, the endogenous E. coli metabolic pathways and genes are engineered to produce increased cytoplasmic concentrations of lactose and / or GDP-fucose compared to the levels found in wild-type E. coli. .. For example, the bacterium comprises at least 10%, 20%, 50%, 2x, 5x, 10x, or higher levels than in the corresponding wild-type bacteria lacking the above genetic modification. In the case of CMP-Neu5Ac, the endogenous Neu5Ac catabolic gene is inactivated and the exogenous CMP-Neu5Ac biosynthetic gene introduced into E. coli produces a cytoplasmic pool of CMP-Neu5Ac not found in wild-type bacteria. .. Methods for producing a pharmaceutical composition comprising purified HMOS include culturing the above bacteria, purifying the HMOS produced by the bacterium, and mixing the HMOS with an excipient or carrier for oral administration. It is done by the stage of producing a dietary supplement. These compositions are useful in methods of preventing or treating intestinal and / or respiratory diseases in infants and adults. Therefore, the composition is administered to a subject who has or is at risk of developing such a disease.

Therefore, the present invention provides a method of increasing intracellular GDP-fucose levels in E. coli by manipulating the organism's endogenous colanate biosynthetic pathway. This is done either in colanic acid precursor biosynthesis or through a number of genetic modifications of the endogenous E. coli gene that are directly involved in the overall regulation of colanate synthetic regulon. The present invention also increases the intracellular concentration of lactose in E. coli with respect to cells grown in the presence of lactose by using the manipulation of the endogenous E. coli gene involved in lactose influx, excretion, and catabolism. I will provide a. In particular, herein describes a method of increasing intracellular lactose levels in E. coli genetically engineered to produce oligosaccharides in human milk by incorporating the lacA mutation into genetically modified E. coli. The lacA mutation prevents the formation of intracellular acetyl lactose, which not only excludes this molecule as a contaminant from subsequent purification, but also eliminates the ability of E. coli to excrete excess lactose from its cytoplasm. Thus, the deliberate manipulation of the E. coli intracellular lactose spool is greatly facilitated.

As used herein, the lac4 gene of the β-galactosidase gene (eg, Kluyveromyces lactis) (eg, by introduction of the functional recombinant Escherichia coli lacZ gene, or from any number of other organisms (eg, Kluyveromyces lactis)). Accumulates intracellular lactose spools while simultaneously retaining low functional levels of cytoplasmic β-galactosidase activity, as provided by GenBank Accession No. M84410 (GI: 173304), incorporated herein by reference). Capable bacterial host cells are also described. Low functional levels of cytoplasmic β-galactosidase are between 0.05 and 200 units, such as 0.05 to 5 units, 0.05 to 4 units, 0.05 to 3 units, or 0.05 to 2 units. (See Miller JH, Laboratory CSH. Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor, NY; 1972, which is incorporated herein by reference for the definition of units) β-galactosidase activity levels. Includes. This low level of cytoplasmic β-galactosidase activity is not high enough to significantly impair the intracellular lactose spool, but nevertheless represents the desired locus during the construction of the host cell background. It is very useful for operations such as type labeling, for the detection of cell lysis due to unwanted bacterial pledge contamination in the fermentation process, or for the easy removal of unwanted residual lactose at the end of fermentation.

In one aspect, the human milk oligosaccharide produced by an engineered bacterium containing an exogenous nucleic acid molecule encoding α (1,2) fucosyltransferase is 2'-FL (2'-fucosyl lactose). .. Preferably, the α (1,2) fucosyltransferase utilized is the wcfW gene of Bacteroides fragilis NCTC 9343 of the invention, or the futC gene of Helicobacter pylori 26695, which has not yet been fully characterized. The wbsJ gene of E. coli strain O128: B12, or any other α (1,2) fucosyltransferase that can use lactose as a sugar acceptor substrate, may be utilized for 2'-FL synthesis. In another aspect, the human milk oligosaccharide produced by an engineered bacterium containing an exogenous nucleic acid molecule encoding α (1,3) fucosyltransferase is 3FL (3-fucosyl lactose), a bacterial cell. Contains an exogenous nucleic acid molecule encoding an exogenous α (1,3) fucosyltransferase. Preferably, the bacterial cell is E. coli. Exogenous α (1,3) fucosyltransferases are isolated from, for example, Helicobacter pylori, H. hepatics, H. billis, C. jejuni, or Bacteroides species. In one aspect, exogenous α (1,3) fucosyltransferases are H. hepatics Hh0072, H. pylori 11639 FucTa, or H. pylori UA948 FucTa (eg, GenBank accession number AF194963 (GI: 28436396), Incorporated herein by reference). The present invention also provides a composition comprising Escherichia coli genetically engineered to produce the human milk tetrasaccharide lactodifcotetraose (LDFT). The E. coli in this example contains an exogenous nucleic acid molecule encoding α (1,2) fucosyltransferase and an exogenous nucleic acid molecule encoding α (1,3) fucosyltransferase. In one aspect, E. coli is transformed with a plasmid expressing α (1,2) fucosyltransferase and / or a plasmid expressing α (1,3) fucosyltransferase. In another aspect, E. coli is transformed with a plasmid that expresses both α (1,2) fucosyltransferase and α (1,3) fucosyltransferase. Alternatively, E. coli is transformed by a chromosomal integrater that expresses α (1,2) fucosyltransferase and a chromosomal integrater that expresses α (1,3) fucosyltransferase. Optionally, E. coli is transformed with plasmid pG177.

Human milk oligosaccharide 3'-S3FL (3'-, containing an exogenous sialyl transferase gene encoding α (2,3) sialyl transferase and an exogenous fucosyl transferase encoding α (1,3) fucosyl transferase Compositions comprising bacterial cells producing (sialyll-3-fucosyllactose) are also described herein. Preferably, the bacterial cell is E. coli. The exogenous fucosyltransferase gene is isolated from, for example, Helicobacter pylori, H. hepatics, H. billis, C. jejuni, or Bacteroides species. For example, exogenous fucosyltransferase genes include H. hepatics Hh0072, H. pylori 11639 FucTa, or H. pylori UA948 FucTa. The exogenous sialyltransferase gene utilized for 3'-S3FL production is a gene described for any one of many origins, such as N. meningitidis and N. gonorrhoeae. Can be obtained from. Preferably, the bacterium comprises a GDP-fucose synthetic pathway.

In addition, the bacterium contains a deficient sialic acid catabolic pathway. "Sialic acid catabolic pathway" refers to a series of reactions that result in the degradation of sialic acid, usually controlled and catalyzed by enzymes. An exemplary sialic acid catabolic pathway in E. coli is described herein. In the sialic acid catabolism pathway described herein, sialic acid (Neu5Ac; N-acetylneuraminic acid) is replaced by the enzymes NanA (N-acetylneuraminate lyase) and NanK (N-acetylmannosamine kinase). It is disassembled. For example, defective sialic acid catabolic pathways are endogenous nanA (N-acetylneurite lyase) (eg, GenBank Accession No. D00067 (GI: 216588), incorporated herein by reference) and / or nanK (N). -Manipulated in Escherichia coli through a null mutation in the (acetylmannosamine kinase) gene (eg, GenBank accession number (amino acid) BAE77265 (GI: 85676015)). Other components of sialic acid metabolism are: (nanT) sialic acid transporter; (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6- Phosphoric acid; (GlcN-6-P) Glucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate.

Moreover, bacteria (eg, E. coli) also include the ability to synthesize sialic acid. For example, the bacterium is incorporated herein by reference to exogenous UDP-GlcNAc 2-epimerase (eg, Campylobacter jejuni neuC or equivalent (eg, GenBank Accession Number (amino acid) AAG29921 (GI: 11095585)). ), Neu5Ac synthase (eg, C. jejuni's neuB or equivalent, eg GenBank accession number (amino acid) AAG29920 (GI: 11095584), incorporated herein by reference), and / or CMP-Neu5Ac synthase (eg). , C. jejuni's neuA or equivalent, eg, GenBank accession number (amino acid) ADN91474 (GI: 307748204), incorporated herein by reference), comprising the ability to synthesize sialic acid.

In addition, the bacterium also contains a functional β-galactosidase gene and a functional lactose permease gene. Bacteria containing the characteristics described herein are cultured in the presence of lactose to recover 3'-sialyl-3-fucosyl lactose from the bacteria themselves or from the bacterial culture supernatant.

Mutations in the endogenous cholanoic acid synthesis gene, the functional lacZ gene, the functional lactose permease gene, the exogenous fucosyl transferase gene encoding the α (1,3) fucosyl transferase, and the α (2,3) sialyl transferase. Also provided is a method of producing 3'-sialyl-3-fucosyllactose (3'-S3FL) in gut flora containing the encoding exogenous sialyl transferase gene. In addition, the bacterium contains a defective sialic acid catabolic pathway. For example, bacteria contain defective sialic acid catabolic pathways due to null mutations in the endogenous nanA (N-acetylneurite lyase) and / or nanK (N-acetylmannosamine kinase) genes. Bacteria also include the ability to synthesize sialic acid. For example, bacteria include exogenous UDP-GlcNAc 2-epimerase (eg, C. jejuni neuC or equivalent), Neu5Ac synthase (eg, C. jejuni neuB or equivalent), and / or CMP-Neu5Ac synthetase (eg, C. jejuni neuB or equivalent). For example, it includes the ability to synthesize sialic acid by providing C. jejuni's neuA or equivalent). Bacteria containing the characteristics described herein are cultured in the presence of lactose to recover 3'-sialyl-3-fucosyl lactose from the bacteria themselves or from the bacterial culture supernatant.

By utilizing an inserted recombinant β-galactosidase (eg, lacZ) gene engineered to produce low but detectable levels of β-galactosidase activity, the original β-galactosidase gene is deleted. Also provided are methods of phenotyping loci in host cells that are or are inactivated. Similarly, the invention also utilizes an inserted recombinant β-galactosidase (eg, lacZ) gene engineered to produce low but detectable levels of β-galactosidase activity in its native β. -Also provides a method of depleting residual lactose from bacterial cultures in β-galactosidase-negative host cells in which the galactosidase gene is deleted or inactivated. Finally, the original β-galactosidase gene is deficient by utilizing an inserted recombinant β-galactosidase (eg, lacZ) gene engineered to produce low but detectable levels of β-galactosidase activity. Also provided is a method of detecting bacterial cell lysis in a culture of lost or inactivated β-galactosidase-negative host cells.

The method for purifying fucosylated oligosaccharides produced by the methods described herein is the step of binding fucosylated oligosaccharides from bacterial cell lysates or bacterial cell culture supernatants to a carbon column, and fucosyl. This is done by eluting the oligosaccharides from the column. Purified fucosylated oligosaccharides are produced by the methods described herein.

Optionally, the invention features a vector, eg, a vector containing nucleic acid. The vector can further include one or more regulatory elements, such as heterologous promoters. Regulatory elements can be functionally linked to a protein gene, a fusion protein gene, or a set of genes linked to an operon to express a fusion protein. In yet another aspect, the invention comprises an isolated recombinant cell, eg, a bacterial cell containing the nucleic acid molecule or vector described above. The nucleic acid sequence can optionally be integrated into the genome.

The term "substantially pure" in connection with a given polypeptide, polynucleotide, or oligosaccharide means that the polypeptide, polynucleotide, or oligosaccharide is substantially free of other biological macromolecules. Means. Substantially pure polypeptides, polynucleotides, or oligosaccharides are at least 75% (eg, at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any suitable calibrated standard method, for example by column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC), or HPLC analysis.

The polynucleotides, polypeptides, and oligosaccharides of the invention are purified and / or isolated. Purified is defined as the lack of a degree of sterility that is safe for administration to human subjects, such as infectious or toxic substances. Specifically, as used herein, "isolated" or "purified" nucleic acid molecules, polynucleotides, polypeptides, proteins, or oligosaccharides, if produced by recombinant techniques, etc. It is substantially free of cell material or culture medium, or, when chemically synthesized, substantially free of chemical precursors or other chemicals. For example, the purified HMOS composition is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. Purity is measured by any suitable calibrated standard method, such as column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC), or HPLC analysis. For example, "purified protein" means a protein that has been isolated from other naturally associated proteins, lipids, and nucleic acids. Preferably the protein comprises at least 10, 20, 50, 70, 80, 90, 95, 99-100% of the dry weight of the purified preparation.

By "isolated nucleic acid" is meant a nucleic acid that does not contain a gene adjacent to it in the naturally occurring genome of the organism from which it is derived. The term is, for example, (a) DNA that is part of a naturally occurring genomic DNA molecule, but in the genome of a naturally occurring organism in which both of the nucleic acid sequences adjacent to that part of the molecule are not adjacent; (B) Nucleic acid incorporated into the vector or into the genomic DNA of proto-nuclear or eukaryotic cells so that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) cDNA, genomic fragment. , Individual molecules such as fragments produced by polymerase chain reaction (PCR), or restriction fragments; and (d) hybrid genes, ie, recombinant nucleotide sequences that are part of the gene encoding the fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acid having a chemically altered and / or modified backbone. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide.

When functionally linked to a nucleic acid sequence, "heterologous promoter" means a promoter that does not naturally associate with the nucleic acid sequence.

The terms "expressed" and "overexpressed" are used in some examples where cells useful in the methods herein can naturally express some of the factors that are genetically modified to produce. In this case, it is used to refer to the fact that the addition of a polynucleotide sequence causes overexpression of the factor. That is, under the same conditions, more factors are expressed by altered cells than when expressed by wild-type cells. Similarly, if a cell does not inherently express a factor that is genetically modified to produce, the term used would simply be to "express" the factor because the wild-type cell did not express the factor at all. There will be.

As used herein, the terms "treat" and "treatment" are used to reduce the severity and / or frequency of symptoms, to eliminate symptoms and / or their underlying causes, and / or Means the administration of a substance or formulation to a clinically symptomatological individual suffering from an adverse condition, disorder, or disease to facilitate improvement or correction of injury. The terms "prevent" and "prevention" mean the administration of a substance or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, thus. Concerning the prevention of the occurrence of symptoms and / or their underlying causes.

The present invention comprises a composition comprising human milk oligosaccharides purified from a culture of a recombinant strain of the invention in which HMOS binds to the pathogen and the subject is infected with the pathogen or is at risk of infection by the pathogen. To provide a method of treating, preventing, or reducing the risk of an infection in a subject, including the step of administering the subject. In one aspect, the infection is caused by the Norwalk-like virus or Campylobacter jejuni. The subject is preferably a mammal in need of such treatment. Mammals are, for example, any mammal, such as humans, primates, mice, rats, dogs, cats, cows, horses, or pigs. In a preferred embodiment, the mammal is a human. For example, the composition is an animal diet for companion animals, such as dogs or cats, and poultry or animals grown for food consumption, such as cattle, sheep, pigs, chickens, and goats (eg, pellets, kibbles, etc.). Can be formulated in mash) or animal feed supplements. Preferably, the purified HMOS is formulated into a powder (eg, a prescription powder for larvae or a dietary supplement powder for adults, each of which is mixed with a liquid such as water or juice before being consumed) or tablets. , In the form of capsules, or pastes, or as an ingredient in dairy products such as milk, cream, cheese, yogurt, or kefir, or as an ingredient in any beverage, or as a prebiotic. It is mixed and incorporated into preparations containing live microbial cultures or prebiotic preparations intended to enhance the growth of beneficial microorganisms either in vitro or in vivo. For example, purified sugar (eg, 2'-FL) can be mixed with Bifidobacterium or Lactobacillus in a probiotic nutritional composition (ie, Bifidobacterium is a normal human gut flora. It is a beneficial ingredient and is also known to utilize HMOS for growth).

The terms "effective amount" and "therapeutically effective amount" of a formulation or formulation ingredient mean a non-toxic but sufficient amount of the formulation or ingredient to provide the desired effect.

The transitional term "comprising" is synonymous with the terms "including," "containing," or "characterized by." Or unlimited and does not exclude additional unquoted element or method stages. In contrast, the transitional phrase "consisting of" excludes any element, stage or component not specified in the claims. The phrase "consisting essentially" defines the scope of the claims as the specified material or stage, as well as the material that does not materially affect the "basic novel features" of the claimed invention. Or limit to "stage".

[Invention 1001]
The step of providing a bacterium containing a functional β-galactosidase gene, an exogenous fucosyltransferase gene, a GDP-fucose synthetic pathway, and a functional lactose permease gene;
The step of culturing the bacterium in the presence of lactose; and the step of recovering fucosylated oligosaccharides from the bacterium or from the culture supernatant of the bacterium.
A method of producing fucosylated oligosaccharides in a bacterium, including.
[Invention 1002]
The method of the present invention 1001 in which the β-galactosidase gene comprises the E. coli lacZ gene.
[Invention 1003]
The method of the present invention 1001 in which the β-galactosidase gene is an endogenous β-galactosidase gene or an extrinsic β-galactosidase gene.
[Invention 1004]
The method of 1001 of the present invention, wherein the bacterium accumulates increased intracellular lactose spools and produces low levels of β-galactosidase.
[Invention 1005]
The method of the present invention 1001 wherein the exogenous fucosyltransferase gene encodes α (1,2) fucosyltransferase or α (1,3) fucosyltransferase.
[Invention 1006]
The method of the present invention 1005, wherein the α (1,2) fucosyltransferase gene comprises the Bacteroides fragilis wcfW gene.
[Invention 1007]
The method of the present invention 1005, wherein the α (1,3) fucosyltransferase gene comprises the Helicobacter pylori 26695 futA gene.
[Invention 1008]
The method of the invention 1005, wherein the bacterium comprises both an exogenous fucosyltransferase gene encoding α (1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α (1,3) fucosyltransferase.
[Invention 1009]
The method of 1001 of the present invention, wherein the GDP-fucose synthetic pathway comprises an endogenous enzyme or an exogenous enzyme.
[Invention 1010]
The method of the present invention 1001, wherein the lactose permease gene is an endogenous lactose permease gene or an extrinsic lactose permease gene.
[Invention 1011]
Steps to provide gut flora containing a functional β-galactosidase gene, an exogenous fucosyltransferase gene, mutations in a colan acid synthase gene, and a functional lactose permease gene;
The step of culturing the bacterium in the presence of lactose; and the step of recovering fucosylated oligosaccharides from the bacterium or from the culture supernatant of the bacterium.
A method of producing fucosylated oligosaccharides in a bacterium, including.
[Invention 1012]
The method of the present invention 1011, wherein the β-galactosidase gene comprises the E. coli lacZ gene.
[Invention 1013]
The method of the present invention 1011, wherein the exogenous fucosyltransferase gene encodes α (1,2) fucosyltransferase or α (1,3) fucosyltransferase.
[Invention 1014]
The method of the present invention 1011, wherein the intestinal bacterium comprises Escherichia coli.
[Invention 1015]
The method of the present invention 1014, wherein the cholanoic acid synthesis gene comprises the wcaJ gene.
[Invention 1016]
The method of the present invention 1014, wherein the bacterium further comprises a mutation in the lon gene.
[Invention 1017]
The method of the present invention 1014 comprising a functional wild-type E. coli lacZ ⁺ gene in which the bacterium was inserted into the endogenous lon gene.
[Invention 1018]
The method of the present invention 1014, wherein the endogenous lacZ gene of E. coli is deleted.
[Invention 1019]
The method of the present invention 1014, wherein the bacterium further comprises an exogenous rcsA or rcsB gene.
[Invention 1020]
The method of the present invention 1014, wherein the bacterium further comprises a mutation in the lacA gene.
[Invention 1021]
Functional β-galactosidase gene, extrinsic sialyl-transferase gene, extrinsic fucosyl transferase gene, GDP-fucose synthesis pathway, deficient sialic acid catalysis pathway, sialic acid synthesis ability, and functional lactose permease gene Method for Producing 3'-Sialyl-3-Fucosyl Lactose (3'-S3FL) in Bacteria Containing:
The step of culturing the bacterium in the presence of lactose; and the step of recovering 3'-S3FL from the bacterium or from the culture supernatant of the bacterium.
[Invention 1022]
The method of 1021 of the present invention, wherein the exogenous sialyl-transferase gene encodes α (2,3) sialyl-transferase.
[Invention 1023]
The method of 1021 of the present invention, wherein the exogenous fucosyltransferase gene encodes α (1,3) fucosyltransferase.
[1024 of the present invention]
The method of 1021 of the present invention, wherein the defective sialic acid catabolic pathway comprises a null mutation in the endogenous N-acetylneurite lyase or N-acetylmannosamine kinase gene.
[Invention 1025]
The method of 1021 of the present invention, wherein the sialic acid synthesizing ability comprises an exogenous UDP-GlcNAc 2-epimerase gene, an extrinsic Neu5Ac synthase gene, or an extrinsic CMP-Neu5Ac synthase gene.
[Invention 1026]
Intestines containing functional lacZ gene, extrinsic fucosyltransferase gene, extrinsic sialyltransferase gene, mutations in endogenous cholanoic acid synthesis gene, functional lactose permease gene, defective sialic acid catalytic pathway, and sialic acid synthesis ability Method for Producing 3'-Sialyl-3-Fucosyl Lactose (3'-S3FL) in Endobacteria:
The step of culturing the bacterium in the presence of lactose; and the step of recovering 3'-S3FL from the bacterium or from the culture supernatant of the bacterium.
[Invention 1027]
The method of the present invention 1026, wherein the exogenous fucosyltransferase gene encodes α (1,3) fucosyltransferase.
[Invention 1028]
The method of the present invention 1026, wherein the exogenous sialyltransferase gene encodes an α (2,3) sialyltransferase.
[Invention 1029]
The method of the present invention 1026, wherein the defective sialic acid catabolic pathway comprises a null mutation in the endogenous N-acetylneulamin lyase or N-acetylmannosamine kinase gene.
[Invention 1030]
The method of the present invention 1026, wherein the sialic acid synthesizing ability comprises an exogenous UDP-GlcNAc 2-epimerase gene, an exogenous Neu5Ac synthase gene, or an exogenous CMP-Neu5Ac synthase gene.
[Invention 1031]
The original β-galactosidase gene is deleted or inactivated by utilizing an inserted recombinant β-galactosidase gene that has been engineered to produce low but detectable levels of β-galactosidase activity. A phenotypic marking method for loci in host cells.
[Invention 1032]
The original β-galactosidase gene is deleted or inactivated by utilizing an inserted recombinant β-galactosidase gene that has been engineered to produce low but detectable levels of β-galactosidase activity. A method of depleting residual lactose from a bacterial culture in a β-galactosidase-negative host cell.
[Invention 1033]
The original β-galactosidase gene is deleted or inactivated by utilizing an inserted recombinant β-galactosidase gene that has been engineered to produce low but detectable levels of β-galactosidase activity. A method for detecting bacterial cell lysis in a culture of β-galactosidase-negative host cells.
[Invention 1034]
Produced by the method of the present invention 1001, comprising the step of binding a fucosylated oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant to a carbon column and the step of eluting the fucosylated oligosaccharide from the column. A method for purifying fucosylated oligosaccharides.
[Invention 1035]
An isolated E. coli bacterium containing a defective cholanoic acid synthesis pathway, reduced levels of β-galactosidase activity, and an exogenous fucosyltransferase gene.
[Invention 1036]
Purified fucosylated oligosaccharide produced by the method of the present invention 1001.
[Invention 1037]
Transformation into a bacterial host strain containing a deleted endogenous β-galactosidase gene, a low-activity replacement functional β-galactosidase gene, a GDP-fucose synthetic pathway, a functional lactose permease gene, and a deleted lactose acetyltransferase gene A nucleic acid construct containing the exogenous fucosyl transferase gene.
[Invention 1038]
The nucleic acid construct of the present invention 1037, wherein the exogenous fucosyltransferase gene encodes α (1,2) fucosyltransferase or α (1,3) fucosyltransferase.
Other features and advantages of the present invention will be apparent from the following detailed description and claims regarding preferred embodiments. Unless otherwise stated, all scientific and technological terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. The Genbank and NCBI submissions indicated by the accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific treatises cited herein are incorporated herein by reference. In case of conflict, this specification, including definitions, will prevail. Moreover, materials, methods, and examples are for illustration purposes only and are not intended to be restrictive.

It is a schematic diagram which shows the synthetic pathway of the main neutral fucosyl-oligosaccharide found in human milk. It is a schematic diagram which shows the synthetic pathway of a major sialyl-oligosaccharide found in human milk. It is a schematic diagram explaining the metabolic pathways and changes introduced into them to manipulate 2'-fucosyl lactose (2'-FL) synthesis in E. coli. Specifically, the lactose synthesis pathway and the GDP-fucose synthesis pathway are illustrated. In the GDP-fucose synthetic pathway: manA = phosphomannose isomerase (PMI), manB = phosphomannomutase (PMM), manC = mannose-1-phosphate guanylyltransferase (GMP), gmd = GDP-mannose-4, 6-dehydrase, fcl = GDP-fucose synthase (GFS), and ΔwcaJ = mutant UDP-glucose lipid carrier transferase. It is a photograph of a thin layer chromatogram of purified 2'-FL produced in E. coli. It is a schematic diagram explaining the metabolic pathways and changes introduced into them to manipulate 3'-sialyl lactose (3'-SL) synthesis in E. coli. Abbreviations are: (Neu5Ac) N-acetylneuraminic acid, sialic acid; (nanT) sialic acid transporter; (ΔnanA) mutant N-acetylneuraminate lyase; (ManNAc) N-acetylmannosamine; (ΔnanK) Mutant N-acetylmannosamine kinase; (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6- P) Glucosamine-6-phosphate; (Fruc-6-P) Fructose-6-phosphate; (neuA) CMP-N-acetylneuraminate synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; And (neuB) N-acetylneuraminate synthase. It is a schematic diagram explaining the metabolic pathways and changes introduced into them to manipulate 3-fucosyl lactos (3-FL) synthesis in E. coli. It is a plasmid map of pG175 expressing the Escherichia coli α (1,2) fucosyltransferase gene wbsJ. In a Western blot photograph of a lysate of E. coli containing pG175 and expressing wbsJ, and a lysate of cells containing pG171, a pG175-inducible plasmid that has the H. pyrroli 26695 futC gene in place of wbsJ and expresses futC. is there. It is a photograph of a thin layer chromatogram of 3FL produced in Escherichia coli containing the plasmid pG176 and induced for the expression of the H. pyrroli 26695α (1,3) fucosyltransferase gene futA by tryptophan addition. A plasmid map of pG177 containing both the H. pylori 26695α (1,2) fucosyltransferase gene futC and the H. pylori 26695α (1,3) fucosyltransferase gene futA, which are configured as operons. 2'-FL, 3FL, and LDFT (lactodif) produced in E. coli, indicated by the plasmids pG171, pG175 (2'-FL), pG176 (3FL), and pG177 (LDFT, 2'-FL and 3FL). It is a photograph of a thin layer chromatogram of Escherichia coli). It is a figure which shows the substitution of the lon gene in Escherichia coli strain E390 with the DNA fragment which has both the kanamycin resistance gene (derived from transposon Tn5) and the wild-type Escherichia coli lacZ + coding sequence. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15). Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-1. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-2. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-3. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-4. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-5. Annotated DNA sequence (in GenBank format) for DNA insertion into the lon region illustrated in Figure 12 (SEQ ID NO: 9-15), a continuation of Figure 13-6. It is a table containing the genotypes of some E. coli strains of the present invention. This is a plasmid map of pG186 expressing the α (1,2) fucosyltransferase gene futC in an operon together with the cholate pathway transcription activation gene rcsB. Escherichia coli containing pG180, which is a pG175-inducing plasmid that has the B. flagiris wcfW gene in place of wbsJ and expresses wcfW, and pG175-inducible plasmid that has the H. pyrroli 26695 futC gene in place of wbsJ and expresses futC. It is a photograph of a Western blot of a lysate of a cell containing a certain pG171. It is a photograph of a thin layer chromatogram of 2'-FL produced in E. coli by cells carrying the plasmid pG180 or pG171 and inducing the expression of wcfW or futC, respectively. It is a photograph of a thin-layer chromatogram showing the kinetics and degree of 2'-FL production in a 10 L bioreactor of E. coli host strain E403 transformed with plasmid pG171. Column chromatogram and TLC analysis for separation of 2'-FL samples made in E. coli from lactose impurities on carbon columns. It is a photograph of a thin layer chromatogram showing 3'-SL in a culture medium produced by E. coli strain E547, which contains a bacterial α (2,3) sialyltransferase and plasmids expressing neuA, neuB, and neuC.

Detailed Description of the Invention Human milk glycans, which contain both oligosaccharides (HMOS) and their complex carbohydrates, play a significant role in the protection and development of human infants, and especially in the infant's digestive tract (GI). The milk oligosaccharides found in various mammals are very different and their composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48: 69-86; Newburg DS, 2001 Adv Exp Med Biol, 501: 3-10). Moreover, glycan levels in human milk vary during lactation and widely among individuals (Morrow AL et al., 2004 J Pediatr, 145: 297-303; Chaturvedi P et al., 2001 Glycobiology, 11: 365-372). Until now, a complete study of the role of HMOS has been limited due to the inability to properly characterize and measure these compounds. In recent years, sensitive and reproducible quantitative methods for analyzing both neutral and acidic HMOSs have been developed (Erney, R., Hilty, M., Pickering, L., Ruiz-Palacios, G. ., and Prieto, P. (2001) Adv Exp Med Biol 501, 285-297. Bao, Y., and Newburg, DS (2008) Electrophoresis 29, 2508-2515). Approximately 200 distinct oligosaccharides have been identified in human milk, and the cause of this diversity is a combination of a few simple epitopes (Newburg DS, 1999 Curr Med Chem, 6:117). -127; Ninonuevo M. et al, 2006 J Agric Food Chem, 54: 7471-74801). HMOS contains five monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetylneuraminic acid,). Neu5Ac, NANA). HMOS usually contains two groups according to its chemical structure: Glc, Gal, GlcNAc and Fuc, and contains the same sugar as the neutral compound bound to the lactose (Galβ1-4 Glc) core, often in the same core structure. In addition, it is divided into acidic compounds containing NANA (Charlwood J. et al, 1999 Anal Biochem, 273: 261-277; Martin-Sosa et al, 2003 J Dairy Sci, 86: 52-59; Parkkinen J. and Finne. J., 1987 Methods Enzymol, 138: 289-300; Shen Z. et al, 2001 J Chromatogr A, 921: 315-321). Approximately 70-80% of oligosaccharides in human milk are fucosylated and its synthetic pathway is thought to proceed in a manner similar to the pathway shown in FIG. 1 (Type I and Type II subgroups are different precursors). Starts with a molecule). A smaller proportion of oligosaccharides in human milk is sialylated or fucosylated to sialylate. Figure 2 outlines possible biosynthetic pathways for sialylated (acidic) HMOS, but the actual synthetic pathways in humans have not yet been fully defined.

Interestingly, HMOS as a class remains very efficiently in the passage of infants into the intestinal tract, a function that is poorly transported through the intestinal wall and resistant to enzymatic digestion of the human intestinal tract. (Chaturvedi, P., Warren, CD, Buescher, CR, Pickering, LK & Newburg, DS Adv Exp Med Biol 501, 315-323 (2001)). One result that remains in this way in the intestinal tract is that the HMOS can function as prebiotics, i.e. utilizing the HMOS to serve as a rich carbon source for the growth of commensal microorganisms resident in the intestinal tract. What you can do (Ward, RE, Ninonuevo, M., Mills, DA, Lebrilla, CB, and German, JB (2007) Mol Nutr Food Res 51, 1398-1405). Recently, there has been a rapid increase in interest in the role of diet and dietary probiotic substances in determining the composition of the gut flora and in understanding the relationship between gut flora and human health (Roberfroid, M., Gibson). , GR, Hoyles, L., McCartney, AL, Rastall, R., Rowland, I., Wolvers, D., Watzl, B., Szajewska, H., Stahl, B., Guarner, F., Respondek, F ., Whelan, K., Coxam, V., Davicco, MJ, Leotoing, L., Wittrant, Y., Delzenne, NM, Cani, PD, Neyrinck, AM, and Meheust, A. (2010) Br J Nutr 104 Suppl 2, Sl-63).

Many human milk glycans have structural homology with cell receptors for intestinal pathogens and play a role in pathogen defense by acting as molecular receptors "decoys". For example, the pathogen strain of Campylobacter specifically binds to glycans in human milk containing the H-2 epitope, namely 2'-fucosyl-N-acetyllactosamine or 2'-fucosyllactose (2'-FL). Campylobacter binding and infectivity is inhibited by 2'-FL and other glycans containing this H-2 epitope (Ruiz-Palacios, GM, Cervantes, LE, Ramos, P., Chavez-Munguia, B., and Newburg, DS (2003) J Biol Chem 278, 14112-14120). Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by HMOSes containing 2'-binding fucose moieties. Several major strains of human calicivirus, especially norovirus, also bind to 2'-linked fucosylated glycans, which are inhibited by human milk 2'-linked fucosylated glycans. Consumption of human milk with high levels of these 2'-linked fucosylated oligosaccharides is from moderate to norovirus, Campylobacter, E. coli-related diarrhea ST, and all causes in a cohort of breast-fed children in Mexico. It is associated with a lower risk of severe diarrhea (Newburg DS et al, 2004 Glycobiology, 14: 253-263; Newburg DS et al, 1998 Lancet, 351: 1160-1164). Influenza (Couceiro, JN, Paulson, JC & Baum, LG Virus Res 29, 155-165 (1993)), Parainfluenza (Amonsen, M., Smith, DF, Cummings, RD & Air, GM J Virol 81, 8341- Several pathogens such as 8345 (2007)) and rotovirus (Kuhlenschmidt, TB, Hanafin, WP, Gelberg, HB & Kuhlenschmidt, MS Adv Exp Med Biol 473, 309-317 (1999)) are also their host receptors. It is known to utilize sialylated glycans as. Sialyl-Lewis X epitopes are Helicobacter pylori (Mahdavi, J., Sonden, B., Hurtig, M., Olfat, FO, et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of norovirus (Rydell, GE, Nilsson, J., Used by Rodriguez-Diaz, J., Ruvoen-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).

Studies suggest that human milk glycans can be used as prebiotics and antibacterial anti-adhesive substances, but the difficulty and cost of producing the right amount of these substances of quality suitable for human consumption. Limits its full-scale testing and recognized usefulness. What is needed is a suitable method for producing a sufficient amount of suitable glycans at a reasonable cost. Prior to the present invention described herein, several individual synthetic approaches have been attempted to synthesize glycans. New chemical approaches can synthesize oligosaccharides (Flowers, HM Methods Enzymol 50, 93-121 (1978); Seeberger, PH Chem Commun (Camb) 1115-1121 (2003)), but the reactions of these methods The thing is expensive and probably toxic (Koeller, KM & Wong, CH Chem Rev 100, 4465-4494 (2000)). Enzymes expressed from engineered organisms (Albermann, C, Piepersberg, W. & Wehmeier, UF Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C, et al. Glycoconj J 16, 205-212 (1999); Johnson, KF Glycoconj J 16, 141-146 (1999); Palcic, MM Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, EJ Curr Opin Chem Biol 4, 110-119 (2000)) provides accurate and efficient synthesis (Palcic, MM Curr Opin Biotechnol 10, 616-624 (1999)); Crout, DH & Vic , G. Curr Opin Chem Biol 2, 98-111 (1998)), the high cost of reactants, especially sugar nucleotides, limits their usefulness for low-cost, large-scale production. Microorganisms have been genetically engineered to express the glycosyltransferases required to synthesize oligosaccharides from the innate nucleotide sugar pool of bacteria (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo , T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179 -183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)). However, the low overall product yield and high process complexity limit the commercial usefulness of these approaches.

Prior to the present invention described herein, which allows for the inexpensive production of large quantities of neutral and acidic HMOS, the ability of this class of molecules to inhibit pathogen binding may be fully investigated or potentially. It was not possible to actually investigate its full range of further functions.

Prior to the present invention described herein, chemical synthesis of HMOS was possible, but was limited by stereospecificity issues, precursor availability, product impurities, and overall high cost. (Flowers, HM Methods Enzymol 50, 93-121 (1978); Seeberger, PH Chem Commun (Camb) 1115-1121 (2003); Koeller, KM & Wong, CH Chem Rev 100, 4465-4494 (2000)). Similarly, prior to the invention described herein, in vitro enzymatic synthesis was similarly possible, but limited due to the need for expensive nucleotide sugar precursors. The present invention overcomes the shortcomings of these previous attempts by providing a novel strategy for inexpensively producing large amounts of human milk oligosaccharides for use as dietary supplements. The invention described herein is Escherichia coli, an engineered bacterium engineered to produce 2'-FL, 3FL, LDFT, or sialylated fucosyl-oligosaccharides at commercially feasible levels. Utilizing (or other bacteria), for example, the method described herein can produce 2'-fucosylated lactose in a bioreactor at> 50 g / L.

Example 1. Manipulation of E. coli to generate host strains for fucosylated human milk oligosaccharide production E. coli K12 protozoal W3110 was selected as the parent background for fucosylated HMOS biosynthesis. This strain has already been modified at the ampC locus with the introduction of the tryptophan-induced P _trpB -cI + repressor construct (McCoy, J. & Lavallie, E. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [ et al.] (2001)) , induction by tryptophan millimolar (Mieschendahl, M., Petri, T. & Hanggi, U. Nature Biotechnology 4, 802-808 (1986) through), recombinant from phage .lambda.P _L promoter Allows economic production of type proteins (Sanger, F., Coulson, AR, Hong, GF, Hill, DF & Petersen, GB J Mol Biol 162, 729-773 (1982)). Strain GI724, an E. coli W3110 derivative containing a tryptophan-induced P _trpB- cI + repressor construct in ampC, was used as the basis for further E. coli strain manipulation (Fig. 14).

Biosynthesis of fucosylated HMOS requires the generation of enhanced cell pools of both lactose and GDP-fucose (Fig. 3). This enhancement was performed in GI724 strain by λRed recombination (Court, DL, Sawitzke, JA & Thomason, LC Annu Rev Genet 36, 361-388 (2002)) and normal introduction of P1 phage (Thomason, L.C., Costantino, N). It was obtained by some manipulation of chromosomes using. & Court, DL Mol Biol Chapter 1, Unit 1.17 (2007)). FIG. 14 is a table showing the genotypes of several E. coli strains constructed for the present invention. The ability of E. coli host strains to accumulate intracellular lactose was first engineered in strain E183 (FIG. 14) by simultaneously deleting the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lacI). .. During the construction of this deletion in GI724 to make El83, the lacIq promoter was placed just upstream of the lactose permease gene lacY. Thus, the modified strain retains its ability to transport lactose from the culture medium (by LacY), but lacks the wild-type copy of the lacZ (β-galactosidase) gene responsible for lactose catabolism. Therefore, culturing the modified strain in the presence of exogenous lactose produces intracellular lactose spools.

Next, the ability of the host E. coli strain to synthesize the extracellular capsular polysaccharide, glucose, was demonstrated in strain E183 with UDP-glucose lipid carrier transferase (Stevenson, G., Andrianopoulos, K., Hobbs, M. & Reeves, It was abolished in strain E205 (Fig. 14) by deletion of the wcaJ gene encoding PR J Bacteriol 178, 4885-4893 (1996)). In the wcaJ null background, GDP-fucose accumulates in the cytoplasm of E. coli (Dumon, C, Priem, B., Martin, SL, Heyraud, A., et al. Glycoconj J 18, 465-474 (2001)). ..

By transducing P1, the thyA (thymidine synthase) mutation was introduced into strain E205 to prepare strain E214 (Fig. 14). In the absence of exogenous thymidine, the thyA strain is unable to make DNA and dies. Defects can be complemented by trans by feeding the wild-type thyA gene into a multicopy plasmid (Belfort, M., Maley, GF & Maley, F. Proceedings of the National Academy of Sciences 80, 1858 (1983)). ). This complement is used herein as a means of plasmid maintenance (maintaining plasmid copy number eliminates the need for conventional antibiotic selection schemes).

One strategy for GDP-fucose production is to enhance the natural synthetic capacity of bacterial cells. For example, this is an enhancement obtained by inactivating enzymes involved in GDP-fucose consumption and / or overexpressing RcsA, a positive regulatory protein in the colanic acid (fucose-containing exopolysaccharide) synthetic pathway. Is. Overall, this metabolic manipulation strategy redirects the flow of GDP-fucose, the end point of cholanoic acid synthesis, to oligosaccharide synthesis (Figure 3). The "GDP-fucose synthetic pathway" is a series of reactions that result in the synthesis of GDP-fucose, usually controlled and catalyzed by enzymes. An exemplary GDP-fucose synthetic pathway in E. coli as described in FIG. 3 is described below. In the GDP-fucose synthesis pathway described below, the enzymes for GDP-fucose synthesis are: 1) manA = phosphomannose isomerase (PMI), 2) manB = phosphomannomutase (PMM), 3) manC = mannose- 1-guanyryl phosphate transferase (GMP), 4) gmd = GDP-mannose-4,6-dehydrase (GMD), 5) fcl = GDP-fucose synthase (GFS), and 6) ΔwcaJ = mutant UDP-glucose Includes lipid carrier transferase.

Specifically, the size of the cytoplasmic GDP-fucose pool in strain E214 is RscA (Gottesman, S. & Stout, V. Mol Microbiol 5, 1599-1606), a positive transcriptional regulator of E. coli for colanic acid biosynthesis. It is enhanced by overexpressing 1991)). This overexpression of RcsA is obtained by incorporating the wild-type rcsA gene containing its promoter region into a multicopy plasmid vector and transforming the vector into an E. coli host, such as E214. This vector typically also has additional genes, particularly one or two fucosyltransferase genes under the control of the pL promoter, as well as thyA and β-lactamase genes for plasmid selection and maintenance. pG175 (SEQ ID NO: 1 and Figure 7), pG176 (SEQ ID NO: 2), pG177 (SEQ ID NO: 3 and Figure 10), pG171 (SEQ ID NO: 5), and pG180 (SEQ ID NO: 6). ) Are all examples of fucosyltransferase expression vectors, each of which also has one copy of the rcsA gene, for the purpose of increasing the intracellular GDP-fucose pool of E. coli hosts transformed with these plasmids. Further positive regulators of cholanoic acid biosynthesis, namely RcsB (Gupte G, Woodward C, Stout V. Isolation and characterization of rcsb mutations that affect colanic acid capsule synthesis in Escherichia coli K-12. J Bacteriol 1997, Jul; 179 ( Similarly, overexpression of 13): 4328-35) can be used in place of or in addition to RcsA overexpression to increase intracellular GDP-fucose levels. Overexpression of rcsB is also obtained by including this gene in a multicopy expression vector. pG186 is such a vector (SEQ ID NO: 8 and Figure 15). pG186 expresses rcsB in the operon with futC under the control of the pL promoter. The plasmid is also driven by its own promoter to express rcsA. pG186 is a derivative of pG175 in which the α (1,2) FT (wbsJ) sequence is replaced by the H. pylori futC gene (FutC is MYC-tagged at its C-terminus). In addition, the E. coli rcsB gene is inserted at the XhoI restriction site immediately 3'of the futC CDS and completed by the ribosome binding site at the 5'end of the rcsB CDS so that futC and rcsB form an operon.

A third means for increasing intracellular GDP-fucose pool may be used as well. Colanic acid biosynthesis increases after introducing a null mutation into the E. coli lon gene. Lon is an ATP-dependent intracellular protease responsible for RcsA degradation, mentioned earlier as a positive transcriptional regulator of cholanoic acid biosynthesis in E. coli (Gottesman, S. & Stout, V. Mol Microbiol 5, 1599- 1606 (1991)). In a lon null background, RcsA is stabilized, RcsA levels are increased, genes responsible for GDP-fucose synthesis in E. coli are upregulated, and intracellular GDP-fucose concentration is enhanced. The lon gene was almost completely deleted and replaced with the functional wild-type but promoter-less E. coli lacZ ⁺ gene (Δlon :: (kan, lacZ ⁺ )) inserted in strain E214 to replace strain E390. Was produced. It was constructed using the λRed recombination operation. FIG. 12 shows a novel composition of a gene engineered at the lon locus in E390. FIG. 13 represents the complete DNA sequence of the annotated region in GenBank format. The genomic DNA sequence around the LacZ + insertion site in the lon region of E. coli strain E390 is described below (SEQ ID NO: 7).

The lon mutation in E390 increases the intracellular level of RcsA and enhances the intracellular GDP-fucose pool. The inserted lacZ ⁺ cassette not only knocks out the lon, but also transforms the lacZ ^- host back to the LacZ ⁺ genotype and phenotype. The modified strain yielded minimal (still easily detectable) levels of β-galactosidase activity (1-2 units), which had a very small effect on lactose consumption during production execution. Although not, it is useful for removing residual lactose at the end of execution, is an easily gradeable phenotypic marker for transferring lon mutations to other lacZ ^- E. coli strains by P1 transduction, and in bioreactors. It can be used as a simple test for cell lysis (eg, caused by unwanted bacteriophage contamination) during production execution.

The production host strain E390 incorporates all of the above genetic modifications and has the following genotypes:
ampC :: (P _trpB λcI ⁺ ), P _lacI ^q (ΔlacI-lacZ) ₁₅₈ lacY ⁺ , ΔwcaJ, thyA ₇₄₈ :: Tn10, Δlon :: (kan, lacZ ⁺ )

A further modification of E390 useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of 2'-FL) is the incorporation of the lacA mutation. LacA is a lactose acetyltransferase that is active only when high levels of lactose accumulate in the E. coli cytoplasm. High intracellular osmolality (eg, caused by high intracellular lactose spools) can inhibit bacterial growth, so E. coli uses LacA to "tag" excess intracellular lactose with an acetyl group. After that, it has evolved a mechanism to protect itself from the high intracellular osmolality caused by lactose by actively excreting acetyl-lactose from the cells (Danchin, A. Bioessays 31, 769). -773 (2009)). Therefore, it is not desirable for acetyl-lactose to be produced in E. coli engineered to produce 2'-FL or other human milk oligosaccharides; this reduces overall yield. Moreover, acetyl-lactose is a by-product that complicates oligosaccharide purification schemes. Incorporation of the lacA mutation solves these problems. Strain E403 (Fig. 14) is a derivative of E390 that has a deletion of the lacA gene and is thus unable to synthesize acetyl-lactose.

The production host strain E403 incorporates all of the above genetic modifications and has the following genotypes:
ampC :: (P _trpB λcl ⁺ ), P _lacI ^q (ΔlacI-lacZ) ₁₅₈ lacY ⁺ , ΔwcaJ, thyA ₇₄₈ :: Tn10, Δlon :: (kan, lacZ ⁺ ) ΔlacA

Example 2. Small-scale 2'-FL production When various alternative α (1,2) fucosyltransferases can utilize lactose as a sugar acceptor and express it in E. coli E214, E390, or E403 under appropriate culture conditions, 2 '-Available for FL synthesis purposes. For example, the plasmid pG175 (ColE1, thyA +, bla +, P _L2- wbsJ, rcsA +) (SEQ ID NO: 1, Figure 7) carries the wbsJα (1,2) fucosyl transferase gene of E. coli strain O128: B12 and is E. coli. The production of 2'-FL can be directed in strain E403. In another example, the plasmid pG171 (ColE1, thyA +, bla +, P _L2- futC, rcsA +) (SEQ ID NO: 5) was found in H. pylori 26695. futCα (1,2) fucosyltransferase gene (Wang, G. , Rasko, DA, Sherburne, R. & Taylor, DE Mol Microbiol 31, 1265-1274 (1999)) and will also direct the production of 2'-FL in strain E403. In a preferred example, the plasmid pG180 (ColE1, thyA +, bla +, P _L2- wcfW, rcsA +) (SEQ ID NO: 6) is the previously uncharacterized Bacteroides fragilis NCTC 9343 wcfWα (1,2) of the present invention. It has a fucosyltransferase gene and directs the production of 2'-FL in E. coli strain E403.

Add tryptophan to the lactose-containing medium of any one culture of strain E214, E390, or E403 transformed with any one of the plasmids pG171, pG175, or pG180 and each particular strain / plasmid combination regard, occur activation of host E. coli tryptophan use repressor TrpR, then suppressed and a decrease in cytoplasmic cI level P _trpB occurs, thereby derepression of P _L, respectively futC, wbsJ or wcfW expression, and 2 ' -FL production occurs. FIG. 8 is a Coomassie blue-stained SDS PAGE gel of E. coli containing pG175 and expressing wbsJ, and lysates of cells containing pG171 and expressing futC. Prominent staining protein band of molecular weight approximately 35 kDa is, after induction P _L observed for both WbsJ and FutC with (i.e., tryptophan after addition) 4 and 6 hours. FIG. 16 is a Coomassie blue-stained SDS PAGE gel of E. coli containing pG180 and expressing wcfW, and lysates of cells containing pG171 and expressing H. pylori futC. P _L after induction (ie, after the addition of tryptophan in the growth medium) for 4 hours and 6 hours, for both WcfW and FutC, prominent staining band was observed at a molecular weight of approximately 40 kDa.

Initial exponential growth of strains in selective media lacking both thymidine and tryptophan (eg, M9 salt, 0.5% glucose, 0.4% casamino acid) for 2'-FL production in small experimental cultures (<100 ml). It was grown at 30 ° C until the phase. And lactose was added to a final concentration of 0.5 or 1% with tryptophan (200 [mu] M final concentration) to induce expression of the driven by alpha (1, 2) fucosyltransferase by P _L promoter. At the end of the induction period (~ 24 hours), TLC analysis was performed on aliquots of cell-free culture medium or thermal extracts of cells (treatment at 98 ° C. for 10 minutes to release the sugar contained within the cells). FIG. 11 shows a TLC analysis of a cytoplasmic extract of engineered E. coli cells transformed with pG175 or pG171. Cells were induced to express wbsJ or futC, respectively, and grown in the presence of lactose. Production of 2'-FL can be clearly observed in thermal extracts of cells carrying either plasmid. FIG. 17 shows a TLC analysis of a cytoplasmic extract of engineered E. coli cells transformed with pG180 or pG171. Cells were induced to express wcfW or futC, respectively, and grown in the presence of lactose. Production of 2'-FL can be clearly observed for both plasmids. Prior to the present invention, the wcfW gene has never been shown to encode a protein with proven α (1,2) fucosyltransferase activity or to utilize lactose as a sugar acceptor substrate.

The DNA sequence (protein coding sequence) of the Bacteroides fragilis strain NCTC 9343 wcfW gene is described below (SEQ ID NO: 4).

Example 3. Production of 2'-FL in bioreactor
2'-FL can be produced in a bioreactor by any one of the host E. coli strains E214, E390, or E403 transformed with any one of the plasmids pG171, pG175, or pG180. Growth of transformants is carried out in a bioreactor with a working volume of 10 L in minimal medium under the control of dissolved oxygen, pH, lactose substrate, defoamer and nutrient levels. A minimal "FERM" medium is used in the bioreactor, which is described in detail below.
Ferm (10 liters): Minimal medium containing:
40g (NH ₄ ) ₂ HPO ₄
lOOg KH ₂ PO ₄
lOg MgSO ₄ .7H ₂ O
40g NaOH
Trace elements:
1.3g NTA
0.5g FeSO ₄ .7H ₂ O
0.09g MnCl ₂ .4H ₂ O
0.09g ZnSO ₄ .7H ₂ O
O.Olg CoCl ₂ .6H ₂ O
O.Olg CuCl ₂ .2H ₂ O
O.O2g H ₃ BO _{3
O.Olg Na 2 MoO 4 .2H 2 O} (pH 6.8)
Add water to make 10 liters
DF204 Defoamer (O.lml / L)
Glycerol 0.99 g 90% glycerol -1% MgSO ₄ -1 × trace elements in fed-batch mode after the (first batch growth), giving the different times at different speeds.

Producing cell densities A ₆₀₀ > 100 are routinely obtained in these bioreactor runs. Briefly, small bacterial cultures are grown overnight in "FERM" in the absence of either antibiotics or exogenous thymidine. Overnight culture (at ~ 2 A ₆₀₀ ) was used to inoculate the bioreactor (10 L working volume, including “FERM”) to an initial cell density of ~ 0.2 A ₆₀₀ . After glycerol prepared in batch mode at 30 ° C. The (A ₆₀₀ to 20) biomass until exhausted, to start the fed batch phase by using glycerol as a finite carbon source. In A ₆₀₀ to 30, to induce 0.2 g / L-tryptophan was added to alpha (1, 2) fucosyltransferase synthesis. The lactose of the first bolus is also added at this point. After 5 hours, a continuous slow supply of lactose is started in parallel with the supply of glycerol. These conditions are continued for 48 hours (2'-FL production phase). At the end of this period, both lactose and glycerol supplies are terminated and residual glycerol and lactose are consumed during the final fermentation period prior to recovery. 2'-FL accumulates at 30-fold higher concentrations in the consumed fermentation medium than in the cytoplasm. The specific yield in the consumed medium varies from 10 to 50 g / L depending on the exact growth and induction conditions. FIG. 18 shows TLC of culture medium samples taken from the bioreactor at various times during 2'-FL production using plasmid pG171 transformed into strain E403. All lactose input was converted to product by the end of the run, with a product yield of approximately 25 g / L 2'-FL.

Example 4.2 Purification of fucosyllactose 2'-FL purification from E. coli fermented broth is carried out through five steps.

1. 1. Clarified Fermented broth is collected and cells are removed by sedimentation in a preparative centrifuge at 6000 xg for 30 minutes. Each bioreactor run yields approximately 5-7 L of partially clarified supernatant. The clarified supernatant has a brown / orange color due to the fractionation of caramelized sugar produced during the fermentation process, especially due to side reactions promoted by ammonium ions present in the fermentation medium.

2. Capturing products with coarse carbon Equilibrate a column (dimensions 5 cm in diameter x 60 cm in length) filled with coarse carbon (Calgon 12 x 40 TR) with a volume of ~ 1000 ml with 1 column volume (CV) of water. Then, the clarified culture supernatant is loaded at a flow rate of 40 ml / min. This column has a total volume of about 120 g of sugar (lactose). After loading and trapping sugar, after washing the column with 1.5 CV of water, 50% ethanol or 25% isopropanol (at this stage, a low concentration of ethanol (25-30%) is sufficient to elute the product). It elutes with 2.5 CV (which can be). This solvent elution step releases about 95% of the total bound sugar on the column and a small amount of color (caramel). In this first stage, the main purpose is to capture the maximum amount of sugar. Separation of contaminants is not the goal. The column can be regenerated by washing with 5 CV of water.

3. 3. A 2.5 L amount of ethanol or isopropanol eluate from the evaporation capture column is rotary evaporated at 56 ° C. to make sugar syrup water (which is typically yellow-brown). Alternative methods that can be used for this step include lyophilization or spray drying.

Four. Flash chromatography on fine carbon and ion exchange media
A column (GE Healthcare HiScale50 / 40, 5 × 40 cm, maximum pressure 20 bar) connected to the Biotage Isolera One FLASH Chromatography System is filled with 750 ml of a 1: 1 mixture of Darco activated carbon G60 (100 mesh): celite 535 (coarse). (Both column fillers were obtained from Sigma). Equilibrate the column with 5 CV of water and load the sugar from step 3 (10-50 g depending on the ratio of 2'-FL to mixed lactose) using either a Celite loading cartridge or direct injection. .. A column is connected to an Evaporative Light Scattering (ELSD) detector to detect peaks of sugars that elute during chromatography. To separate 2'-FL from monosaccharides (if present), lactose, and colorants, perform a 4-step gradient of isopropanol, ethanol, or methanol, for example for B = ethanol: step 1, 2.5 CV 0%. B; Stage 2, 4 CV 10% B (eluting monosaccharides and lactose contaminants); Stage 3, 4 CV 25% B (eluting 2'-FL); Stage 4, 5 CV 50% B (some Elute the colorant and partially regenerate the column). Further column regeneration is performed with 50% methanol and 50% isopropanol. Fractions corresponding to sugar peaks are automatically collected in 120 mL bottles, pooled and directed to stage 5. In certain purification runs from longer than normal fermentations, passing the 2'-FL-containing fraction through anion-exchange and cation-exchange columns can remove excess protein / DNA / caramel contaminants. Resins tested and successful for this purpose are Dowex 22 and Toyopearl Mono-Q for anion exchange resins and Dowex 88 for cation exchange resins. Mixed bed Dowex resins have proven unsuitable because they tend to adsorb sugars with high affinity through hydrophobic interactions. FIG. 19 illustrates the performance of a 1: 1 mixture of Darco G60: celite in the separation of lactose from 2'-fucosyl lactose when used in flash chromatography mode.

Five. Evaporation / freeze-drying
Rotary evaporate 3.0 L of 25% B solvent fraction at 56 ° C until dry. The solid sugar mass is dissolved in the smallest amount of water, the solution is frozen and then lyophilized. At the end of the process, a white, crystalline sweet powder (2'-FL) is obtained. The purity of the resulting 2'-FL is between 95 and 99%.

Sugars are routinely analyzed for purity by impregnating a 1 μl aliquot on an aluminum-lined silica G60 thin-layer chromatography plate (10 × 20 cm; Macherey-Nagel). Mixtures of LDFT (Rf = 0.18), 2'-FL (Rf = 0.24), lactose (Rf = 0.30), trehalose (Rf = 0.32), acetyl-lactose (Rf = 0.39) and fucose (Rf = 0.48) (each) Simultaneously test with 5 g / L concentration for sugar as a standard substance. Unfold the plate in 50% butanol: 25% acetic acid: 25% aqueous solvent until the tip is within 1 cm of the top. Improved sugar separation can be obtained by performing two consecutive runs and drying the plate between runs. The sugar spot is sprayed with a sulfuric acid-ethanol solution of α-naphthol (83% (v / v) ethanol, 2.4 g of α-naphthol in 10.5% (v / v) sulfuric acid), and at 120 ° C. for several minutes. Visualize by heating stage. High molecular weight contaminants (DNA, protein, caramel) remain at the starting point or form smears of Rf below LDFT.

Example 5.3 Transforming any one of the FL-producing E. coli host strains E214, E390, or E403 with a plasmid expressing α (1,3) fucosyl transferase that can use lactose as the sugar acceptor substrate results in humans. It will produce a milk oligosaccharide product, 3-fucosyl lactose (3FL). FIG. 9 illustrates the pathways utilized in the engineered E. coli strains of the invention to obtain 3FL production. For example, the plasmid pG176 (ColE1, thyA +, bla +, P _L2- futA, rcsA +) (SEQ ID NO: 2) has the α (1,2) FT (wbsJ) sequence replaced by the Helicobacter pylori futA gene. (Dumon, C, Bosso, C, Utille, JP, Heyraud, A. & Samain, E. Chembiochem 7, 359-365 (2006)). Transformation of pG176 with any one of the host E. coli strains E214, E390, or E403 would direct the production of 3FL. FIG. 11 shows a TLC analysis of 3FL production of E403 transformed with pG176. In addition, some other related bacterial α (1,3) fucosyltransferases identified in Helicobacter pylori that can be used to direct the synthesis of 3FLs, such as "11639 FucTa" (Ge, Z.,, Chan, NW, Palcic, MM & Taylor, DE J Biol Chem 272, 21357-21363 (1997); Martin, SL, Edbrooke, MR, Hodgman, TC, van den Eijnden, DH & Bird, MI J Biol Chem 272 , 21349-21356 (1997)) and "UA948 FucTa" (Rasko, DA, Wang, G., Palcic, MM & Taylor, DE J Biol Chem 275, 4988-4994 (2000)). In addition to the α (1,3) fucosyltransferase of H. pylori, the α (1,3) fucosyltransferase (Hh0072, sequence accession AAP76669) isolated from Helicobacter hepatics is a non-sialylated and sialylated type. 2 It is active against both oligosaccharide acceptor substrates (Zhang, L., Lau, K., Cheng, J., Yu, H., et al. Glycobiology (2010)). In addition, there are several additional bacterial α (1,3) -fucosyltransferases that can be used to make 3FLs according to the methods of the invention. For example, closely related homologues of Hh0072 are found in HH Billis (HRAG_01092 gene, sequence accession number EEO24035), and C. jejuni (C1336_000250319 gene, sequence accession number EFC31050).

3FL biosynthesis utilizes small scale or control of dissolved oxygen, pH, lactose substrate, defoamers, and carbon: nitrogen balance in culture tubes and flasks, as previously described for 2'-FL. Performed in a bioreactor (10 L working volume). Cell density A ₆₀₀ to 100 can be obtained in the bioreactors, specific 3FL yield of up to 3 g / L is obtained. Approximately half of the 3FL produced is found in the culture supernatant and half is found intracellularly. Purification of 3FL from E. coli culture supernatant is obtained using almost the same technique as described above for 2'-FL. The only substantial difference is that 3FL elutes from the carbon column at lower alcohol concentrations than 2'-FL.

Example 6. Simultaneous production of human milk oligosaccharides 2'-fucosyl lactose (2'-FL), 3-fucosyl lactose (3FL), and lactodifucohexaose (LDFT) in E. coli E. coli strains E214, E390, and E403 were first introduced. As discussed, accumulating both lactose and GDP-fucose cytoplasmic pools and transforming them with a plasmid expressing either α (1,2) fucosyl transferase or α (1,3) fucosyl transferase, respectively, respectively. Human lactose oligosaccharides 2'-FL or 3FL can be synthesized. The tetrasaccharide lactodifucotetrose (LDFT) is another major fucosylated oligosaccharide found in human milk, α (1,2)-and α (1,3) -linked fucose residues. Including both. pG177 (FIG. 10, SEQ ID NO: 3) shows that the wbsJ gene is a two-gene operon containing the Helicobacter pylori futA gene and the Helicobacter pylori futC gene (ie, α (1,3)-and α (1,2)). -A derivative of pG175 that has been replaced by an operon that contains both fucosyltransferase. E. coli strains E214, E390, and E403 are transformed with plasmid pG177 and produce LDFT when grown on a small scale or in a bioreactor as described above. In FIG. 11 (lane pG177), LDFT produced in E. coli, indicated by pG177, was observed in the analysis of cell extracts by thin layer chromatography.

Example 7. 3'-SL Synthesis in E. Coli Cytoplasm The first step in 3'-sialyl lactose (3'-SL) production in E. coli is a host background that accumulates a cytoplasmic pool of both lactose and CMP-Neu5Ac (CMP-sialic acid). The generation of strains. Cytoplasmic lactose accumulation is obtained by growth in the presence of lactose and inactivation of the endogenous E. coli β-galactosidase gene (lacZ), taking care to minimize the polar effect on the lac permease lacY. .. This accumulation of lactose spools has already been obtained in E. coli hosts engineered to produce 2'-FL, 3FL, and LDFT and is described above.

Specifically, methods known in the art (eg, Ringenberg, M., Lichtensteiger, C. & Vimr, E. Glycobiology 11, 533-539 (2001); Fierfort, N. & Samain, E. J Biotechnol 134). , 261-265 (2008)), and the scheme for generating the cytoplasmic CMP-Neu5Ac pool is shown in FIG. In this scheme, the E. coli K12 sialic acid catabolic pathway is first disrupted by introducing null mutations into the endogenous nanA (N-acetylneurite lyase) and nanK (N-acetylmannosamine kinase) genes. "Sialic acid catabolic pathway" refers to a series of reactions that result in the degradation of sialic acid, usually controlled and catalyzed by enzymes. An exemplary sialic acid catabolic pathway in E. coli is shown in FIG. In the sialic acid catabolic pathway in FIG. 5, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminate lyase) and NanK (N-acetylmannosamine kinase). Other abbreviations for the sialic acid catabolic pathway in FIG. 5 are (nanT) sialic acid transporter; (ΔnanA) mutant N-acetylneuraminate lyase; (ΔnanK) mutant N-acetylmannosamine kinase; (ManNAc-6). -P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P); N-acetylglucosamine-6-phosphate; (GlcN-6-P) glucosamine-6-phosphate; (Fruc- 6-P) Fructose-6-phosphate; (neuA) CMP-N-acetylneuraminate synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; and (neuB) N-acetylneuraminate synthase Including.

Next, E. coli K12 introduces the ability to synthesize sialic acid by providing three recombinant enzymes due to its lack of a denovosial acid synthesis pathway; UDP-GlcNAc-2-epimerase (eg, neuC), Neu5Ac synthase (eg, neuC). For example, neuB), and CMP-Neu5Ac synthetase (eg, neuA). Equivalent genes from C. jejuni, Escherichia coli K1, Haemophilus influenzae, or Neisseria meningitidis can be utilized (compatiblely) for this purpose.

Next, to make 3'-sialyl lactose, the addition of sialic acid to the 3'position of lactose was performed using bacterial α (2,3) sialyltransferase, Neisseria gonorrhoeae and Neisseria gonorrhoeae. Numerous candidate genes have been described, including those of (Gilbert, M., Watson, D.C., Cunningham, AM, Jennings, MP, et al. J Biol Chem 271, 28271-28276 (1996); Gilbert, M., Cunningham, AM, Watson, D.C., Martin, A., et al. Eur J Biochem 249, 187-194 (1997)). The Neisseria enzyme is already known to use lactose as the acceptor sugar. Recombinant meningitis enzymes produce 3'-sialyl lactose in engineered E. coli (Fierfort, N. & Samain, E. J Biotechnol 134, 261-265 (2008)). Figure 20 shows E. coli strain E547 (ampC :: (P _trpB λcI ⁺ ), P _lacI ^q (ΔlacI-lacZ) ₁₅₈ ) with plasmids expressing neuA, B, C, and bacterial α (2,3) sialyl transferase. The TLC analysis of the culture medium obtained from the culture of lacY ⁺ , ΔlacA, Δnan) is shown. The presence of 3'-sialyl lactose (3'-SL) in the culture medium is clearly observed.

Example 8. Production of human milk oligosaccharide 3'-sialyl-3-fucosyl lactouse (3'-S3FL) in E. coli Prior to the present invention described herein, any particular fucosyltransferase gene and any particular fucosyltransferase gene in the same bacterial strain. It was not expected that 3'-S3FL could be produced by combining specific sialyltransferase genes. The following describes the results demonstrating that the combination of the fucosyltransferase gene and the sialyltransferase gene in the same LacZ ⁺ E. coli strain caused the production of 3'-S3FL. These unexpected results may be due to the surprisingly loose substrate specificity of the particular fucosyltransferase and sialyltransferase enzymes utilized.

Humans utilize different combinations of 6 α (1,3) fucosyltransferases and 6 α (2,3) sialyltransferases encoded in the human genome to synthesize sialyl-Lewis X epitopes (de Vries, T. et al. , Knegtel, RM, Holmes, EH & Macher, BA Glycobiology 11, 119R-128R (2001); Taniguchi, A. Curr Drug Targets 9, 310-316 (2008)). Not only do these sugar transferases differ in their tissue expression patterns, but they also differ in their acceptor specificity. For example, human bone marrow α (1,3) fucosyltransferase (FUT IV) fucosylates acceptors based on type 2 (Galβ1-> 4 Glc / GlcNAc) chains only if they are not sialylated. There will be. In contrast, "plasma-type" α (1,3) fucosyltransferases (FUT VIs) utilize type 2 acceptors and are found indiscriminately in the mammary gland and kidney, whether or not they are sialylated. Type (promiscuous) "Lewis" α (1,3/4) fucosyltransferase (FUT III) is sialylated and non-sialylated Type 1 (Galβ1-> 3 GlcNAc) and Type 2 acceptors (Easton, EW, Schiphorst, WE) , van Drunen, E., van der Schoot, CE & van den Eijnden, DH Blood 81, 2978-2986 (1993)). A similar situation exists for the human α (2,3) sialyltransferase family, in which different enzymes show significant differences in acceptor specificity (Legaigneur, P., Breton, C, El Battari, A., Guillemot, J. C, et al. J Biol Chem 276, 21608-21617 (2001); Jeanneau, C, Chazalet, V., Auge, C, Soumpasis, DM, et al. J Biol Chem 279, 13461-13468 (2004)). This diversity of acceptor specificity is an important issue in the synthesis of 3'-sialyl-3-fucosyl lactose (3'-S3FL) in E. coli, that is, it can act cooperatively to synthesize 3'-S3FL. It highlights an important issue in identifying suitable combinations of fucosyl- and sialyl-transferase (using lactose as the initial acceptor sugar). However, since human and all other eukaryotic fucosyl and sialyl-transferases are secretory proteins located in the Golgi apparatus, they are the task of 3'-S3FL biosynthesis in the bacterial cytoplasm. Not very suitable for.

Some bacterial pathogens are known to incorporate fucosylated and / or sialylated sugars into their cellular envelope, typically for host mimicry and immune evasion reasons. For example, Neisseria meningitidis and Campylobacter jejuni can incorporate sialic acid into the galactose moiety in their capsule lipooligosaccharide (LOS) through a 2,3-bond (Tsai, CM, Kao, G. & Zhu, P). . I Infection and Immunity 70, 407 (2002); Gilbert, M., Brisson, JR, Karwaski, MF, Michniewicz, J., et al. J Biol Chem 275, 3896-3906 (2000)), some of the E. coli Strain incorporates α (1,2) capsule group into lipopolysaccharide (LPS) (Li, M., Liu, XW, Shao, J., Shen, J., et al. Biochemistry 47, 378-387 (2008); Li, M., Shen, J., Liu, X., Shao, J., et al. Biochemistry 47, 11590-11597 (2008)). Strains with Helicobacter pylori can not only incorporate α (2,3) -sialyl groups, but also α (1,2), α (1,3), and α (1,4) -fucosyl groups. Can be incorporated into LPS and thus show a wide range of human Lewis-type epitopes on its cell surface (Moran, AP Carbohydr Res 343, 1952-1965 (2008)). Most bacterial sialyl and fucosyltransferases act in the cytoplasm, i.e., they are more suitable than eukaryotic Golgi localized sugar transferases for the methods described herein.

E. coli strains engineered to express the above transferase accumulate a cytoplasmic pool of lactose and an additional pool of either the nucleotide sugar GDP-fucose or the nucleotide sugar CMP-Neu5Ac (CMP-sialic acid). To do. Addition of these sugars to the lactose acceptor was performed in these engineered hosts with candidate recombinant α (1,3) -fucosyl or α (2,3) -sialyltransferase and 3-fucosyl lactose. And 3'-sialyl lactose, respectively. Finally, the two synthetic abilities are combined into one E. coli to produce 3'-S3FL.

E. coli strains have been developed that accumulate both lactose and GDP-fucose cytoplasmic pools. This strain, when transformed with a plasmid that overexpresses α (1,2) fucosyltransferase, produces 2'-fucosyllactose (2'-FL) at levels of ~ 10-50 g / L bacterial culture medium. To do. Substitution of this host's α (1,2) fucosyltransferase with the appropriate α (1,3) fucosyltransferase produces 3-fucosyllactose (3FL). Bacterial α (1,3) fucosyltransferase then acts with bacterial α (2,3) sialyltransferase to produce the desired product, 3'-S3FL.

Α (1,3) fucosyltransferase (Hh0072) isolated from Helicobacter hepatics is active against both non-sialylated and sialylated type 2 oligosaccharide acceptor substrates (Zhang, L., Lau, K). ., Cheng, J., Yu, H., et al. Glycobiology (2010)). To measure the utilization of lactose acceptors, this enzyme is cloned, expressed, and evaluated to assess 3FL production in the context of current GDP-fucose-producing E. coli hosts. Hh0072 is also examined with various bacterial α (2,3) sialyl transferases for its competence in 3'-S3FL synthesis. As an alternative to Hh0072, two characterized homologous bacterial type 3-fucosyltransferases identified in Helicobacter pylori, ie "11639 FucTa" (Ge, Z., Chan, NW, Palcic, MM & Taylor, DE J Biol Chem 272, 21357-21363 (1997); Martin, SL, Edbrooke, MR, Hodgman, TC, van den Eijnden, DH & Bird, MI J Biol Chem 272, 21349-21356 (1997)), and " UA948 FucTa ”(Rasko, DA, Wang, G., Palcic, MM & Taylor, DE J Biol Chem 275, 4988-4994 (2000)) exists. These two paralogs show different acceptor specificity, where "11639 FucTa" utilizes only type 2 acceptors and is a strict α (1,3) fucosyltransferase, whereas "UA948 FucTa" is loose. It has acceptor specificity (utilizes both type 1 and type 2 acceptors) and is capable of producing both α (1,3) -and α (1,4) -fucosyl bonds. Determining the exact molecular basis for this difference in specificity (Ma, B., Lau, LH, Palcic, MM, Hazes, B. & Taylor, DE J Biol Chem 280, 36848-36856 (2005)) The characterization of several additional α (1,3) fucosyltransferase paralogs from a variety of additional H. pyrrolis strains revealed that there was a significant variety of acceptor specificities among the strains.

In addition to enzymes from H. pylori and H. hepatics, other bacterial α (1,3) -fucosyltransferases are optionally used. For example, closely related homologues of Hh0072 are found in H. bilith (HRAG_01092 gene, sequence accession number EEO24035), and C. jejuni (C1336_000250319 gene, sequence accession number EFC31050).

The 3'-S3FL synthesis in E. coli is described below. The first step towards this is to combine one E. coli strain with the ability to synthesize 3-fucosyl lactose, as outlined above, and the ability to produce 3'-sialyl lactose, also outlined earlier. Introducing all of the chromosomal gene modifications discussed above into a new host strain would simultaneously accumulate a cytoplasmic pool of three specific precursors: lactose, GDP-fucose, and CMP-Neu5Ac. This "complex" strain background is then used to drive with two multicopy plasmids that are compatible with gene expression, or by placing both enzyme genes on the same plasmid as the artificial operon, α (1). , 3) Simultaneous host production of fucosyltransferase and α (2,3) sialyltransferase. Some acceptor specificities of bacterial α (1,3) fucosyltransferase and α (2,3) sialyltransferase, especially with respect to fucosylation of 3'-sialyllactose and sialylation of 3-fucosyllactose, and α (1). , 3) Evaluate different combinations of fucosyltransferase and α (2,3) sialyltransferase. Calibration levels and ratios of 3'-SL, 3FL, and 3'-S3FL are monitored, for example by TLC, identified by NMR, calibrated mass spectrometry utilizing specific ion monitoring, or capillary electricity. Accurate quantification by either migration (Bao, Y., Zhu, L. & Newburg, DS Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis. Anal Biochem 370, 206-214 (2007)).

The sequences corresponding to SEQ ID NOs described herein are provided below.

The sequence of PG175 is described below (SEQ ID NO: 1):

The sequence of pG176 is described below (SEQ ID NO: 2):

The sequence of pG177 is described below (SEQ ID NO: 3):

The sequence of Bacteroides fragilis NCTC 9343 wcfW CDS DNA is described below (SEQ ID NO: 4):

The sequence of pG171 is described below (SEQ ID NO: 5):

The sequence of pG180 is described below (SEQ ID NO: 6):

The sequence of W3110Δlon :: Kan :: lacZ with RBS E. coli strain K-12 substrain W3110 is described below (SEQ ID NO: 7):

The sequence of pG186 is described below (SEQ ID NO: 8):

Other Aspects The present invention has been described with a detailed description thereof, but the above description illustrates and is not intended to limit the scope of the invention as defined by the appended claims. .. Other aspects, advantages, and changes are within the scope of the following claims.

The patents and scientific treatises referred to herein establish knowledge available to those of skill in the art. All US patents cited herein and US patent applications published or not yet published are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. The GenBank and NCBI submissions indicated by the accession number are incorporated herein by reference. All other published references, documents, manuscripts, and scientific treatises cited herein are incorporated herein by reference.

Although the present invention has been specifically shown and described in connection with its preferred embodiments, various changes may be made in shape and details, which are also covered by the appended claims. It will be understood by those skilled in the art that it is included in.

Claims (28)

(i) Mutations in the lacA gene, and
(ii) An isolated E. coli bacterium that is unable to synthesize acetyl lactose, including the exogenous lactose-accepting fucosyltransferase gene. The isolated Escherichia coli bacterium according to claim 1, wherein the mutation in the lacA gene prevents the formation of intracellular acetyl lactose. The isolated Escherichia coli bacterium according to claim 1 or 2, further comprising a defective cholanoic acid synthesis pathway. The isolated Escherichia coli bacterium according to claim 3, wherein the defective colanic acid synthesis pathway comprises a mutation in the colanate synthesis gene. The isolated Escherichia coli bacterium according to claim 4, wherein the cholanoic acid synthesis gene comprises the wcaJ, wzxC, wcaD, wza, wzb, or wzc gene. The isolated Escherichia coli bacterium according to any one of claims 1 to 5, further comprising an inactivated mutation in the lon gene. The isolated Escherichia coli bacterium according to claim 6, further comprising a wild-type Escherichia coli lacZ ⁺ gene having no functional promoter inserted into the lon gene. The isolated Escherichia coli bacterium according to any one of claims 1 to 7, further comprising an exogenous Escherichia coli rcsA or Escherichia coli rcsB gene. Either of claims 1-8, wherein the exogenous lactose-accepting fucosyl transferase gene comprises the Bacteroides fragilis wcfW gene or the Helicobacter pylori 26695 futA gene. The isolated Helicobacter pylori described. The invention according to any one of claims 1 to 8, which comprises both an exogenous fucosyltransferase gene encoding α (1,2) fucosyltransferase and an exogenous fucosyltransferase gene encoding α (1,3) fucosyltransferase. Isolated E. coli bacterium. The isolated Escherichia coli bacterium according to any one of claims 1 to 10, further comprising a lactose permease gene. The isolated Escherichia coli bacterium according to claim 11, wherein the lactose permease gene contains an endogenous lacY gene. The isolated Escherichia coli bacterium according to claim 11, wherein the lactose permease gene contains an exogenous lactose permiase gene. The isolated Escherichia coli bacterium according to any one of claims 1 to 13, further comprising an exogenous sialyltransferase gene. The isolated Escherichia coli bacterium according to claim 14, wherein the exogenous sialyltransferase gene encodes α (2,3) sialyltransferase. The defect sialic acid catabolic pathway further comprising a null mutation in the endogenous N-acetylneurite lyase gene or a null mutation in the endogenous N-acetylmannosamine kinase gene, according to any of claims 1-15. Isolated E. coli bacterium. The isolated E. coli bacterium according to any one of claims 1 to 16, further comprising a functional β-galactosidase gene containing a detectable reduced level of β-galactosidase activity as compared to that of a wild-type E. coli bacterium. .. The isolated Escherichia coli bacterium according to claim 17, wherein the reduced level of β-galactosidase activity comprises between 0.05 and 200 units. 17. Isolation according to claim 17, wherein the reduced level of β-galactosidase activity comprises between 0.05 and 5 units, between 0.05 and 4 units, between 0.05 and 3 units, or between 0.05 and 2 units. Escherichia coli bacteria. The isolated Escherichia coli bacterium according to claim 17, further comprising a deletion of the endogenous β-galactosidase gene. The isolated Escherichia coli bacterium according to claim 17, wherein the functional β-galactosidase gene comprises an exogenous functional β-galactosidase gene. The isolated E. coli bacterium according to any one of claims 1 to 21, which accumulates increased cytoplasmic lactose levels and the increased intracellular lactose levels are at least twice as high as those in the corresponding wild-type bacterium. The isolated Escherichia coli bacterium according to any one of claims 1 to 22, further comprising a plasmid expression vector. It further contains and increases the (i) lactose permease gene, (ii) reduced levels of β-galactosidase activity compared to that of wild E. coli bacteria, and (iii) guanosindiphosphate (GDP) -fucose synthetic pathway. The isolated Escherichia coli bacterium according to any one of claims 1 to 23, wherein the intracellular lactose level is accumulated and the increased intracellular lactose level is at least 20% higher than the level in the corresponding wild-type bacterium. The isolated Escherichia coli bacterium according to any one of claims 1 to 24, which is cultured in the presence of lactose in a bioreactor. The isolated Escherichia coli bacterium according to any one of claims 1 to 25 for producing fucosylated oligosaccharides. The isolated Escherichia coli bacterium according to claim 26, wherein the fucosylated oligosaccharide comprises 2'-fucosyllactose, 3-fucosyllactose, or lactodifucotetraose. The isolated Escherichia coli bacterium according to claim 27, wherein the fucosylated oligosaccharide comprises 2'-fucosyl lactouse (2'-FL).

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